SG192023A1 - Radiation cross-linked collagen gel, and preparation method and usage method thereof - Google Patents

Abstract

36RADIATION CROSS-LINKED COLLAGEN GEL, AND PREPARATION METHOD AND USAGE METHOD THEREOFAbstractThe present invention relates to radiation cross-linked collagen gel, and a preparation method and usage method thereof. To this end, the present invention comprises a cross-linked collagen material made by irradiating liquid collagen with radioactive rays,10 wherein the concentration of said collagen is specifically 0.1-10% (W/V), and the radiation dose (dose rate X time) is 0.1 - 40 kGy on the basis of 1 kGy/hr. The present invention configured as above can prepare a formulated collagen gel using a physical cross-linking15 method instead of a chemical cross-linking method, specifically carries out the formulation by mixing biocompatible materials, and provides a method capable of using a cross-linked collagen hydrogel in wound dressings, graft materials, cell cultures and the like.20 Therefore, the present invention provides an industrially convenient and safe preparation method, thereby instilling a good image to a customer by greatly improving the quality and confidence in the products.25 (Fig. 1

Description

RADIATION CROSS-LINKED COLLAGEN GEL, AND PREPARATION

METHOD AND USAGE METHOD THERECH

Technical Fleld

The present disclosure relates to a radiation- crosslinked collagen gel and the preparation method and use thereof, and more particularly to a preparation metnod which can prepare a formulated collagen gel using a physical crosslinking method in place of a chemical crosslinking method. In the present disclosure, collagen is formulated with & biccompatible material and crosslinked to provide a collagen hydrogel which can be used for wound dressings, graft materials and cell culture. Accordingly, the present disclosure provides an 1H industrially convenient and safe method for preparing a collagen gel which has significantly improved quality and confidence, and thus presents a good image to

CONSUMES.

Background Art

As 1s well known, collagen is a structural protein which forms soft tissues SUCH a5 dermis, tendon/ ligament, blood vessels and the likes, and hard tissues such as bones, and accounts for about 1/3 of

Z% total protein in mammals. 20 or more types of collagen are known, and about 90% thereof 1s type I collagen which forms the skin, tenden/ligament, bones and the like.

Collagen is a triple-stranded molecule having a molecular weight of about 300,000 dalton (100,000 dalton for each strand) and is composed of -GAY- repeats, wherein G is glycine (the smallest amino acid), and X and ¥ are amino acids other than glycine.

Collagen ig currently used in medical applications, including hemostatic agents, wound dressings, artificial blood vessels, materials for reducing wrinkles, eta. in the case of hemostatic agents, Avitene which 1s a collagen powder extracted from calf skin was first developed in 1374 and has been 5 used Lo date.

Collagen has various advantages, including low antigenicity, high blocompatibility and bio- absorbability, induction of cell adhesion, growth and differentiation, blood coagulation, hemostatic affects, and compatibility with other polymers.

However, products made only of collagen have Low physical properties (tensile strenath, elasticity, degradability, etc.), and thus are somewhat difficult zo use in applications that reguive physical properties. 1% For this reason, c¢ollagen products have been produced by adding cther biocompatible materials {synthetic polymers, Dioproteins, carpohydrates and other compounds) having sufficient tensile sirength or tear strength.

In addition, c¢ollagen products are formulated with chemical compcunds to improve the physical properties thereof, but most of the compounds used are harmful to the human body.

Prigcry art documents related to collagen products includes Korean Patent Registration No GE3T765R {Application No. 2006-01289466; entitled “Method for preparing water-solulrle oligopeptides from porcine skin collagen by irradiation with radiation”).

Specifically, Korean Fatent Registration Ne. 08378LE discloses a method for preparing water-soluble oligopeptides from porcine skin collagen by irradiation with radizazticn, the method comprising the steps of: irradiating porcine skin collagen with y-rvays at a dose of 50-300 kGy, and dissolving the irradiated porcine skin collagen in a 5-10 fold amount (W/V; of 0.05-0.1M

Nall solution to form a water-soluble collagen; and adding 0.5-1 wti of papain enzyme fo the water-soluble collagen, allowing the mixture to stand for 0.5-4 hours, and fractionating the enzyme-Treated material into specific ocligopeptides having molecular welghts of 16,000-5,000 dalton, 5,000-3,000 dalton and 3,000-2,000 dalton, respectively.

The above-mentioned prior art will now De described in further detail.

The ster of making porcine skin collagen walter- soluble comprises: a process of removing unnecessary materials (fats and impurities) from the porcine skin and washing the remaining porcine skin with water; a ib preocess of cutting the washed porcine skin to a suitable size and crushing the cut porcine skin with ulirasonic waves; and a process of lrradiating the crushed porcine skin with radiation at a dose of 50-300 Gy and dissolving the irradiated porcine skin in a 5-10-fold amount (W/V) of 0§.10M NaCl sclution, preferably 0.05- 0.1% NaCl =olution. Herein, a suitable radiation dose

Tor making the porcine skin collagen is 100 kGy or more,

An existing method requires the use of large amounts of acidic and alkaline chemical materials, wheresas the 25% above-mentioned prior art can produce water-soluble low- molecular-weight materials at a very high yield (4 times or higher that of the existing method) without having to usa acid or salt). using enzyme

Hydrolvase such as papain is added to the irradiated porcine skin to dissociate the water-soluble component... This step comprises: a process of adding 1.5-

Iowt% of papaln fo the irradiated porcine skin and allowing the mixture to stand for (¢.%-4 hours so as to reduce the molecular welght of the water-soluble component; fracrtionating the low-molecular-weight component aooording te molecular welght using a wltratiloration process; a process of freeze-drying the fractionated peptide components, chereby preparing porcine skin collagen-—derived specific oligopeptides.

However, the above-mentioned prior art alsc has a problem in that, because It 1s the technology of reducing rather than increasing the molecular weight of collagen, it is impossible To Tormalate the cligopeptides alone and to formulate the oligopepitidss with biocompatible materials.

Thus, the anove-menticoned prior art also has a problem in that, because lt reduces the molecular weight of collagen, it cannot provide a hydrogel formulation which can be used for wound dressings, graft materials and cell culture.

As a result, the above-mentioned prior art does not provide a preparation method which 1s industrially wh applicable, convenient and safe.

Detailed Description of the Disclosure Technical Problem

The present disclosure has been made In order fo 25 solve the above-described problems occurring in the prior art, and it 1s a first object of the present disclosures TO crepare A crosslinked collagen by irradiating liguid collagen with radiation. A second obilect of the present disclosure 1s to prepare a formulsted collagen gel using a physical crosslinking method In place of a chemical crosslinking method. A third object of The present disclosure is to provide the use of a cressiinked collagen hydrogel, formulated with a picocompatible material, for wound dressings, graft materials and cell culture. A fourth object of the

= present disclosure is Le provide an industrially convanlent and safe preparation method. & fifth obiect of the present disclosure 1s to provide a crosslinked collagen gel, which has significantly improved quality and confidence, and thus can present a good image to consumers, and Che preparation method and use thereof.

Technical Sclutlion

To achieve the above obtects, tha Dresant disclosure provides a method for preparing a crosslinked collagen gel, the method comprising irradiating liguid collagen with radiation to prepare a crosslinked coliagen.

The present disclosure also provides a method of preparing a crosslinked material by Mixing a biocompatible material and Tiguid collagen and irradiating the mixture with radiation, and a merhnod of using the crosslinked material as a tissues repair material for wound healing, a skin graft, a bone graft or the like.

The present disclosure also provides a method of using a hydrogel of the present disclosure in a& mixture with a cosmetic component, or using the hydrogel as a mask pack in a mixture with a cosmelblc component, or using the hydrogel as a wound dressing.

The present disclosure also provides a method of using a radiation-crossliinked collagen g=l, the method comprising: obtaining a partially crosslinked collacen using a low dose cof radiation: pouring the partially 20 crosslinked collagen together with a gelling solution inte a mold to form a gel; pouring cells and a medium on the gel; culturing the cells on the gel; and using Lhe cultured material (hydrogel) in a mixture with a cosmetic component, or using the hydrogel as a mask pack in a mixture with a cosmetic component, or using the

: hydrogel as a wound dressing, or applying the hydrogel toooan injured skin, or dsing the nydrogel as oa skin graiit material.

The present disclosure also provides a method of 5ousing a radiation-orosslinked collagen gel, the method comprising: obtaining a partially cressiinked collagen using a low dose of radiation; mixing the partially crosslinked collagen with a gelling solution to form a gel; drying the gel to formm a thin film; pouring cells and a medium onto the L£iim; culturing the cells on fhe film; and grafting the cultured corneal cells Into an eyeball.

The present disclosure also provides a method of veing a radiation—crosslinked collagen gel, ithe method ih comprising: obtaining a partially crosslinked collagen veing a low dose of radiation; mixing the partially crosslinked collagen with a gelling solution and ceils; culturing the mixture; finely grinding the cultured material; placing the ground material In a syringes; and grafting the material in the syringe inte ths skin or bone by injection.

Advantageous Bifects

As described above, the present disclosurs 2% provides a method of preparing a orvosslinked collagen by irradiating figuid collagen with radiation.

According to the present disclosure, a formulated collagen cel CAN bye prepared using a physical crosslinking method in place of a chemical crosslinking method.

Particularly, the present disclosure provides a method of using a crosslinked collagen hydrogel, formulated with a biocompatible material, for wound dressings, graft materials and cell culture.

Thus, the present disclosure provides an industrially convenient and safe preparation method.

Due to the above effects, the collagen gel according to the present disclosure hag significantly improved quality and confidence, and thus can present a

Hooogood image Lo consumers.

Brief Description of the Drawings

FLG. 1 ds a schematic view showing crosslinking coliagen using radiation. 16 FICE, 2 to 6 are photographs of radiation- crosslinked collagen gels according to the present disclosure.

FIGS. 7 and 8 are photographs o¢f a hydrogel comprising a partially crosslinked collagen according to 13 the present disclosure.

FIGS. 9 to 11 show the degree of partial crosslinking of collagen as a function of radiation dose in the present disclozure.

FIG. 12 is a photograph of a radiation-crosslinked collagen hydrogel according to the present disclosure.

FIG. 13 is a photograph of a radiation-crosslinked collagen film according fo The present disclosure.

FIGS. 14 te 148 are photographs of particles obtained from a radiation-crosslinked collagen hydrogel 2% according to the present disclosure.

FIGs. 17 to 1Y schematically show methods of mel ling a racdiaticn~crosslinked collagen for cell culture according to the present disclosure.

Mede for Carrying Out the Disclosure

Hereinafter, preferred embodiments for achieving the above effects of the present disclosure will be described in detail with reference to the accompanying drawings.

g

FIGS. 1 to 19 show a crossiinked collagen gel according to Lhe present disclosure ang the preparation method and use thereof. in he following description, 3 detailed description of known functions and coniigurations incorporated herein will be omitted when it may obscure the subject matter of the present disclosure.

Also, the terms used in the following description are terms defined taking into consideration the functions obtained in accordance with the present disclosure, and may be changed in accordance with the option of a producer or a usual practice. Accordingly, definitions of these terms must be based on the overall description herein.

The technology of formulation using radiation oan

Ive explained by crosslinking of free vadicals, and methods for crosslinking ny radiation includes gamma-ray rrradiation, electron ray iLrradiation, X-ray irradiation and the like.

ZU Gamma-rays are short-wavelength rays from cobalt- 00 radioactive isotope and have high penetrability.

Gamma-ravys are used in various applications, including sterilization of products, improvement in the physical proverties of polymers, and coloring of jewels.

Z5 Electron yavys ars electromagnetic waves having high kinetic energy, which are obtained by applying a high voltage te thermal electrons produced from tungsten electrically heated to high femperature. Dlectron rays are ionization energy, Like gamma-rays or X-vays, and 34 can be used In various industrial fields, and the dose therecl can be controlled depending on the intended use.

Irradiation with radiation can be gerformed in the absences of chemical substances without being substantially influenced by Temperature, humidity or pressure, Ir addition, iE is simply used for sterilization and is also cost-effective.

The method for crosslinking by radiation eliminates Lhe reed Lo remove residual Toxic crosslinking agents and can cause chemical reactions even in a solid state or at low temperature, unlike chemical crosslinking methods that use formaldehyde and the like. In addition, it can easily control the physical properties of a material using a controlled dose of yadiagtion without changing the composition of the material. Thus, methods for processing polymers using radiation have been studied and developed in varicus flelds, including hydrogels for wound healing and burn treatment, biomaterials and the like.

Accoraing to the general theory of radiation chemistry, a solution of collagen in a water-soluble anlvent ig crossiinkad by hydroxyl radicals {OH) produced by irradiation with radiation, and collagen can be crosslinked by radiation.

Crosslinking ocours by radical polymerization.

ZU Radical polymerization that 13 a chain rEACTION gensrally progresses in the order of initiation, propagation or growth, fermination and movement . An initiator of radial polymerization is used to produce radicals, and a compound such as peroxide or an azo compound, which is sarily controlled, 13 used as the initiator. In addition to the initiator compound, energy such as heat, light or radiation, is alse used Lo produce radicals, and crosslinking can be performed without using a chemical compound. When radiation 1s irradiated, free radicals are produced by the supply of nergy to a water molecule or a material, The free radicals influence the crosslinking of the material. The degree of crosslinking depends on the dose of radiation, and When produced froe radicals are consumed, crossiinking is terminated, Thus, the degrees of crosslinking of a material can be controlled by controlling the dose of radiation.

A radiation-treated materizl exists as a polymer solution or a hydrogel depending on the degree of crosslinking.

Collagen has the properties of a phase transition polymer, Es used herein, the term “phase transition polymer” refers fo a polymer whose physical properties change in LESponse Lo external stimuli 530 as temperature, pH, electric fields and Light. A polymer whose physical properties change in TESnONS a io temperature 1s referred to as a temperature-sensitive polymer. Particularly, collagen has the properties of the Cemperature-sensitive polymer. Collagen exlsts in a io liguld phase at low temperature and changes to an opaque gel phase when reaching a low critical temperature.

Specifically, at a temperavure lower than the low critical solution temperature (LOS3TY, collagen is dissolved due to a hydrogen bond with water, and the

ZU hydrogen bond 1s broken with increasing temperature so that the polymer units agglomerate with each other to form a gel or precipitate. When the gel is formed, the bond is a physical bond which is not a strong bond such

AS a covalent bond, Dut ig attributable Lo an 25 intermolecular force such as a hydrophobic bond or a polar bond. Generally, in the case of neutralized polymers, the gel is produced by hvdrophobic interaction.

A collagen solution partially crosslinked by radiation also nas the properties of & temperature- sensitive polymer. Particulariy, when the partially crosslinked oollagen solution is peoelvmerized to form a gel, it shows & btransparent/semi-transparent formulation and a relatively high elasticity.

The partially crosslinked collagen solution can be un

Lod used as a tool for cell culture. When a formulation comprising general pure collagen is gelled, It shows an opague IZormulation and low elasticity, In addition, the use of the partially crosslinked collagen solution oan advantageously shortan The gelling time.

The partially crosslinked collagen solution can be used in the tissue engineering field, the cosmetic field and the like. Particularly, it can be used as a scaffold for cell culture In the Tissue engineering field. 0 Specifically, it can be used In a method of culturing cells using a produced collagen gel, a method of culturing cells using a produced collagen film, and a method of culturing cells wsing a gel formed afer mixing Lhe crosslinked collagen With celis,

Particularly, the cultured cells can be used in various applications depending on Che characteristics and

Fen ] mE ane sf the oem Ta won iir hd LE LL LAE ETE LAE Led iNT Aen OD.

A collagen hydrogel obtained by irradiation with radiation is in the form of crystallized collagen. It ig pilocompatible and flexible and can be formulated fo have reguired physical properties. Thus, it is used as bio-

Tissue in various fields, including wound dressing, cosmetic and regenerative medicine fields. A gel formed by physical crosslinking has no chemical crosslinking £5 agent harmful fo the human body, and thus is receiving attention as a medical material. Products produced by this method have advantages in that they are easily produced, are harmless Lo humans and environments, and can be formulated in various forms.

Thus, this polymer 13 a good material that can be used as a drug delivery material, a cell delivery marerial or an injectable extracellular matrix in the tissue engineering fleld.

Radiation-based technology also enables the development of medical devices. When operations to which coilagen-based materials and radiatlion-based devices (e.o., X-ray devices ©r gamma nives) are applied are studied and developed, they will lead to a significant development of fLizsus engineering.

The radiaticon-crossliinked collagen gel will now be described in further detail.

Az shown In FIG. 1, the praesent disclosure is characterized in that a crosslinked collagen is prepared by irradiating liguid collagen with radiation.

The radiation that is used in the present digclosure may De any one selected from gamma-rays, electron ravs ana X-rays.

The concentration of collagen that is used in the present disclosure is preferably 0.1-10% (W/V), and the 1% dose (dose rate = time) of radiation is preferably 0.1- 40 kGy.

If the collagen concentration 1s lower than 0.1%, the degree of crosslinking of collagen will be low so that the partially crossiinked collagen cannot be gelled, and 1f the collagen concentration is higher than 10%, dt will difficult for current technology to mix the collagen 50 as to have a uniform concentration distribution. For this reason, the collagen concentration is preferably 0.1-10% (W/V).

If the dose of radiation is lower than 3.1 kGv, the degree of crosslinking of collagen will be low so that the partially crosslinked coollagen cannot be gelled, and LI Lhe dose of radiation is higher than 440 kGy, collagen will be decomposed rather than being partially crosslinked. For this reason, the dose of radiation is preferably 6.140 kGy.

Meanwhile, a method for preparing the radiatrion- crossiinked collagen gel according to the present disclosure is as follows.

Specifically, Lhe radliation-crossliinked collagen gel can be prepared Dy mixing 3% (W/V of collagen with each of 3% (W/V) of synthetic polymer Pluronic F-127, 1% (W/V) of PEC (polyvethviene oxide (MW. = 100,000) and 3% (W/V) of hydroxyapatite and crosslinking the mixture by irradiation with gamma-rays.

Another method for preparing the radiation- crosslinked collagen gel according to the present disclosure 1s as follows.

Specifically, the radiation-crosslinked collagen 100 gel can be prepared by mixing 3% (W/V) of collagen with each of 3% (W/V) of blopolymer hyaluronic acid (M.W. = 2,000 EK) and 3% (W/V) of chondroitin sulfate and crosslinking the mixture hy irradiation with gamma-rays.

Still another method for preparing the radiation 15% crossiinked collagen gel according to Lhe present disclosure 1s as follows.

Specifically, radiation-crosslinked collagen gel can he prepared Dy mixing 30% (W/V) of collagen with each of 3% (W/V) of silicone (Dow Corning, 77-9800), 6% (W/V) of glycerin and 6% (W/V) of PRS and crosslinking the mixture by irradiation with gamma-rays, in which the

PBS component includes 2.8 mg of sodium phosphate and 7.6 mg of scdium chioride per mL of the final volume.

According to the z2bove methods of the present disclosure, The radiaticn~crosslinked collagen gels shown in FIGS. 2 to © can be obtained.

The above-described configurations of the present disclosures can be medified in various forms.

It is to be understood that the present disclosure 20 is not limited to the specific forms mentioned in the above detailed descriprion and includes all modifications, equivalents and substitutions within the sprit and scope of the pressnt disclosure as defined in the appended claims.

The effects of the radiation-crosslinked collagen gel according to the present disclosure and the preparation method and use thereof are as follows.

According to the present disclosure, a formulated collagen gel CAT be prepared BE iT a physical crosslinking method in place of a chemical <¢rosslinking method. Particularly, a crosslinked collagen gel formulated with a biccompatible material can be used for wound dressings, graft materials and cell culture. Thug, the presant disclosure provides an industrially convenient and safe method for preparing the crosslinked collagen gel.

AL Rnalysis of the degree of crosslinking of 33-10% collagen as a function of radiation dose (5-40 kGy)

Purpose: to analyze physical properties as a function of collagen concentration and gamma-ray dose

Method 1} Each of 3%, 6% and 10% ccllagens is prepared in a container. 23 The collagens are lrradiaved with gamma-rays at doses of 5, 25 and 40 kGy, respectively. 3) The physical properties of the collagens crosslinked under the above conditions are analyzed.

LE) Appearances (transparency and gelling tendency) 25% are visually observed.

Bj} The shrinkage rate (percent decrease in volumo) of nydrogel is examined. ) The gel strengnh (maximum stress) is measured using a measurement instrument. = Instrument: Rheo meter (CR-200DX). = Conditions: measurement item (gel strength}, penetration distance (2.5 mmi, table speed (50 mm/min, and adapter (No. 1 &1bmm).

Dy Degradability is determined as the number of 3h days during which the gel remains in the presence of collagenase. rn TT mony om Orgs me even yy be pe my de em eT wm Fee T Soe IRIN ows Ladenase Concentoatliont uli ma/mL an Poh

Ej The water content {change in water content) of croaalinked onl Tamer fae analvrad Ry comma rioo Fhe wrod ht

Crossiinsed Collagen LE analyzed DRY Comparii Hy Che weidgnt

I oy A ey ae {en dn gh AB Fe sam dpe ey oy AS em Tred om A ee a alter Irecze GIryindg wiih Tne weldht arter snyoeration of the crosslinked collagen.

Results 1 7 Camry ees my tn 7m em pn 1 Sr veer noes le 17 ar 1) Appearance: Transparent gel (having weak vellow at 40 kGvy)

IRS: 2} Shrinkage rate (percent decrease in volume) =

Code nn oh emg ee - A a — omy tp 4 pe ae mp] 4m a ry pra {total welght {gy of gamma ray-lrradiated sample) - . 4 rk ae a TE Soe me debe . yore muy welght (g) of gel mass) / total weight (gg) of gamma rav- 4 ae dE a de a 3 “ 1 irradiated sample ¥X 100

Tan Bem

Takhle 1

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Shrinkage rate | Gamma-ray dose (kGy)

RS I

[I ed eye ios 3 70 3

PLnnoen tration I) 20 ai i i i i

PoE) § | : a Be Rd AAR rrr er es AM AA i meres ero

Pon Laon 7 a . i

I] POLO .8 18.0 { a a rrr ene

HE Foo “on Pom om i

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Concentration ls Pon Can [rs i } : £2) i : ; :

E rE ian a Cann ; ad deh LF POT oe Pada wd i & | 6.0 | 45.2 F357

A ym wim TD Ee oF ye 4) Dedgradablliity foia VE)

DEA Ta be 7 I»)

LAS lalla =

P Degradabl Livy | Gamma-ray dose (Kay) :

Ef en pe me pe md ne [= Pon Poa i

POLONCENT TATION 1 Poa PoE i { | : i i

Poe 1 | | !

LA | ow LEE Dowd :

iG fan [— Dom ow xR Dow wk :

Degradability (days) — % 1 cay or less, ** 2 davs or less, FY days oy less, and FF more than 7 odavs fg Tal omy + pms wm be me de meh my 4 wpm de en ae pn de - _— 2) waver COonTent {cha nge In Wailer content) = (weight (g) of hydrated collagen gel - weight ({(g} of c. oy np fam J comm en Td em Te em md mm Th mo LT crpm dh pri de 4 freeze-dried crosslinked collagen gel) / weight (gg) of oo ts 1 TA mlremed een TT omeres 3 freeve-dried crosslinked collagen gel

Tab Tom A

Ae Chas de RI w

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Watery content | Gamma-ray dose (Gy) :

Hare Cent | Gamma cay dose (by) : i , \ . 1 3 (changes in water | : co : content, fold) ; : erm] i i | i

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PALONUenRU rat IOon J 25 Pal ; ne

LT meses bese eee eer ete eee reeeeeeeeeeereeeee] = orn. Ae CER i 5 PAUL H.3 21.1 i : ~ Poae oe oo FO id 18.8 9.1 HH

TM A i

Faampia 2

BE. Analysis of the degree of crogslinxing of 32-10% / od 3

Toa oan mom om Ea eb Dn ae a Ie LAY eet gelatin as od una TLon OL radiation cose { =A SG Yl

Purpese: to analyze physical properties as a function of gelatin concentration and gamma-ray dose

GE Me TR ee i Method

To TT a am} ow ge So any 3 Oy Ye dem Doe mn pm mg md Lo

Lhowmach oi 23, Er oang 10% Gelatingd 13 prepared In

A gontainer.

TIN FT gem, Tom om we en mn per my od mm dm me md EEE apr I~ ne ay <) lhe Jeilatlinsg are irraclalted wilh gamma-rays at

Amos ed 6 PE FAT ere em td tra Ts

GOoSes GL LH, £0 anda au Kissy, respelUTiveiy. “yn TY TER | nn po po Ge — Go Ge crn TT mm my yn 3 fhe vnysical properties of The collagens mp pra mm Eg ge Lee dy i] en ae my we Yo cm - dn ee ym er em pol crosslinked under the above conditions are analyzed.

Lh Lp ny pe mn en em de pm mpm pm nn - np fren TY on - + - yr

A) Appearances (transparency and gelling tendency) . . ! ~ a 107 \ eo Tem wo 3 are visually observed.

TIE Men embed og bem eres oom bee ry pra ee py Ee Tom epg em 4 oem wre] os ) 21 The shrinkage rate | percent decreass In volume; oo CE ata Lo at £3 OI nyarogel 15 examined. ey i —— rt pe gy vs pepe Be . Gove me ond pm om gn De a: “) ine Gel STraendTi {maximum stress) 15 measured

T wy on bop pv vo Tey om ve pg a pe ey EY IN - Instrument: Rheo meter (CR-500DX) .

£4 Ae A ny pm mm gm on Lo ery 1 A yey py - Conditions: measurement item (gel strength}, ry my nm ed J “3 I gen fom bn pron pn gop era se Wen penetration distance (Z.5 mm), table speed (50 mm/min, pd my od my pre em po TY 1 FT pn and adaptor (No. 1 &1l5mm) ™ Te wom emf en hem a TG de — wd fon Fy 3pm pe eed nm fe pm pm mn yp my pm A >) Degradanplliity 13 determined as he number of = days during which the gelatin remains in the presence of collagenase.

I Toe cy on neg en pn de ge oy de deme wT p 7 em TIT C — Collagenase concentration: 0.1 mg/ml in PRS mo The mn very de oy A . no Dm x yp pa fo pm - LE

El} The water content (change in water content) of crosslinked collagen is analyzed by comparing the welght

Ee my ee pn yp od pep pe vp ge pe i 3m [ 1m ie J Sg gwd pm my de A nem — 1 arter Lrgeze GQUVLIOO Wl Lhe welgnt SELLE ry Sra Lon of the crogslinked collagen.

Results

T 4 To pr gm om my pm en pen mom Fo er om pm gma rm mm my pm mp} 1) Avpoearancs { CYanSumarancy

Tobe tom O

LGiliTS i ; \

Appearance | Gamma-ray dose [(KEy) i prore A ec Trm——— a

Concentration C5 CEE | Ail (53 : : | i 1 wu i i i i i i i i

Lo Cw [I Dok i i : ! i i [a Eo doa Ea Dow i

Lo Powwow Uk d os Dodew ob i = es eee eters emer ih Avpearances (transparency) - F OEUS, ** Semi— fre pen my mmm pny . Ge mn gm pe om + ode Ed oe Fe on ogre je en he transparent, FER Lransoarenc, oR Transparent fveillowish) 2) Bhrinkage rate (percent decrease in volume) = (total weight {ag} obi gamma ray-irradiated sample) - “ye yt np jm ET een oo dem nb or Tu er or ro AT gn Len om weight (gg) of gel mass) / total weight (gy of gamms ray- 2mm mT my ne de ow 7 x TN

Lrradiated sample X 1400

TU ey Tn o

Lakzle © : SHrLnrags rate (OF) | Gamma-ray dose (khy 1 i

Concentration : 5 25 40 : i i ! : : i (i) ; : ; :

LL ! - PL, PL :

P32 Pa 131.8 An Ld : io Poa i - boa ;

Pa Po 22 Pan

Fem reve mam merase mame: re EE Ba RRA A A A A ere se a 1 Gel strength (maximum stress) ah Table 7

TO

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Gel strength (WN) Gamma-ray dose (kKEv) ! ! Concent ral lon | 5 25 40 ; i : 1 - L | ¢ ; (%) i : i 3 i 4.5% | 7.9 7.6 i & : 4,4 | 9.6 | fk ee rere

A ™ Sarid 14 dm ng 4% Uegradaniiity (rays)

Ma ee Je oy lable o

PEG aOanY LITY : ama-Lay ose (Hbys

Concentration = | £5 : 40

Pos Pos I. i ! i 3 : ¥ [I i ES i : 1

LA Pow + Po : i i [I Po = bog i

Tempore od 43a de don nym EF 3 3 mpm he ~ 3 nd om =

Degradabzlizty (days) - * 1 day or iess, ** 2 days i Tom im on wk kT 3 om en on ay mn pn de bey om gm ea or less, J days or less, and **** more than 7 davs 2) Water content {change 1n water content) = (welght {g) of hydrated collagen gel ~ weight {(g) of fa 1 EA ed ue Ak J a fee tnd lin Ld Ta il ef Fam TI ade “A Joi. gd Ad freeze-dried crosslinked collagen gel) / weight (gq) of freeze-dried crosslinked collagen gel 1 te nO

LU LdizLe oF : Hata See Carns —rmy cteveen flu ; i YEoroLun lg Lendl RRL LACRLim TT Lady Al nid LRA J i : {charge in water | i ; : content, fold) i

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Concentration 5 i Zh : 40 5 ! i i i ee edrie ET 1 £4£ £4124£ ee emer wrens

C10 1a Pog Pas

Lxample 3

Fa » fo aon = J - J . - my pdr am Ee . - a — A

CO. Materlal prepared by irradiating a mixture of collagen and & synthetic polymer with radiation

Purpose: To examine whether a mixture of collagen apd a synthetic polymer can be formulated.

BA md Te em od pleat hod 3 I ror four ~~ TT a re my <m = od wow de hey fe “2 vpn 3 a } 3% { WH / Vo) COL LAden LE miXed WIth eaaonh oy SWE E

. [=RE Wm Tae Dd 1% < Tye “ mmr Tm I. (w/v) Pluronic F-127, 1% (w/v) PEC {polyethylene oxide nA td TO OO pe md HO fen fer FE [ER ty py (4 SW = 100 pM} ana 2% {w FAY J DYAroxyapd Cite, snr Then war enor er 1d py deemed ber FE YRTITL Sem TS LF AD FL mm ed IIT Leroy

CITOSslinred DY damna-ravs { SOAnG 2 Kis Yo.

DR Th on hm rpm dem ny py om A . i SS ye en en A en eo od

Z} ine 1 Ig VEIT properties On The DTOSS LL nKed a oy deny ey mm ny re mixture are analyzed. on J Arne ATANCES FrANanaren oy vy cre TT 1 or emmy 3 yr

HS ADDearancaes Transparency and dei llng enc ancy are visually observed,

Tile om em A . “a ~ - I I I pe a 4

B) The shrinkage rate (percent decrease in volume) of the mixture 13 examined,

LU C } Ine gel sTrendg i {max imum stress) LE Measured

Using a measurement instrument.

Tome dyer or 10) wy yyiem oom pe Or ET = LTIST ramen. wed meter {(CR- S500 DEY. - Conditions: measurement item {gel strength), on o oh a G3 ex dy en mem “i o ry doo Te i pn men od {OE vm Are em meno tration distanges {2.5 mm; Pp Callie Spead (DU WMm/ming

TA op od Ime 1 4 PFT Eman ih and adaptor (No. I S&iommy.

DU) Degradability is determined as the number of days during which the crosslinked mixture remains in the presence of collagenase. ~ Collagenase concentration: 0.1 ma/mi. in PRE 24 FE} The water content (change in water content; of = ma — Tq en Leen Fey ea dower Pe Toor a] = od FE epee np em bem

Che Crossiinged mLIXLUDe LSE analyzed Dy comparing Tne weight after freeze drying with the weight after hydration of the crosslinked mixture.

Feasulits as 1Y Appearances (tyansparency)

Pn Re 3 Toy

Table 10

Ese

Appearance C Synthetic polymer mixed with collagen er amv eee i ee £5 2 mm met eet ee = een mtr orb HABA 18 A tee : . . bo . | !

Gamma-ray dose | Hydroxyapatite PEG (MUW.=100FY | Pluronio Fe

Tod | i [a ; i lw — i [ox {Hap i i : 5 ; : | od Ea i | distribution) i | i i i

Poon bo | wo HE !

Appearance {(Uransparency) - fF opacue, fF Seni - nransparent, rE transparent, ok transparent {veellowish) 5 30 . Ym pm hy bey yen oo deg en - Ln prem pn Dam wren Tomy em y — 2) Shrinkage rate (percent decrease in volume) = v

Se de eT " vm pg fe, de 5 -, HT mg mn : y Pd ram pnd om 3 on Toes (total welght {gg} of gamma ray-irradiated samp. e) - weight (gr of gel mass) / total weight {g} of gamma ray- en ge on ed qm be ed omer 1a - Ea prradlated sample ALG

Taye 10

Lalbie Ld

D Shrinkage rate (percent | Synthetic polymer mixed with collagen decreases Ln volume) : i eo]

Gamma-ray dose (Gy) Hydroxyapatite PPEO (MLW =:00K) LD Pluronio : : Po SE ‘ :

Cog inn PN Laon

Fo Pua Pol Poa i ew fuein ha it YC EE FAB 51818 61301851 2 1 meek eet mes oe 5m nk mm 21 342.28 £328 2 5 ok et 3 meee bare 1255813 370 05 5005s

Pal Po oak Poll

IZ

2) Gel strength (maximum stress)

To be wT C2

Lamle LZ

CGel strength (maximum | Synthetic polymer mixed with oollagen : i 5 :

Patrass) (MN) ! ; me eee eee err —————— ee eo ot A tm wt

Gamma-ray dose (kGy) Hydroxyapatite CRED (MLW. =I100K) 1 Pluronic ° pT | | ; : ! | PE-ieT

P11. | 40.3 | 3.0 a : la EE /i I me pe od my bm 4 TS de nr fern) 4 pedradanilloy Gays

LU lanle 13

Degradabilivy (dave) Synrhatic polymer mixed with collagen ee ee emt mm eee smssnmnpni as instore anit ran rot gr ee visser cnsconcnd

Gamma~ra YOOnEe (kis Yl PMY Or OEY aDAT ITE Vom EAL (14 CWT 00K J ELTON

F177 o Poe ok HE [oe Po Pox - yp - on <7 9 El J ovr * i 3 . om en gm “3 a mow te

Degradability (days) - * 1 day or less, ** 2 days _ 3 on demo 7 lev vro I = er = md i A pe pm dey “3 Joy wpe or oLess, rr days or less, and *YY*YY more than 7 days £00 TT om Am omy a pm 3 vor A “ a + pe 59 Water content (change In water content) a my pg J de - 47 md en om en J yo J 4 J oO ~ 4 {weight (gg) of hydrated collagen gel - weight (ag) of

Lo freeze-dried crosslinked collagen gel) / weight (gg) of = ey de 3 im a BS gn in 1 k on - S frecze~dried crozslinked collagen ge:

Table 14

Water content (change | Synthetic polymer mixed with collagen i ’ bo © }

Lin water content, fold) | {

Gamma-ray dose (Hay) PHYOIONYAarat ine PEG {MLE = LU0E] Po Piuronio re rs sre io BEER Poll od 40.0

EF [mmm 2. Material prepared by lrradiating a mixture of collagen and a bicpolymer with radiation = Purpose: to examine whether a mixture of collagen and a blopolymer can be formulated.

Method

Iv 2% collagen is mixed with each of 3% hyaluronic acid (M.W., = 2,000 Ky) and 2% chondroitin sulfate and then crosslinked by gamma-zavs (5 and 25 ky). 27 The physical properties of the crosslinked mixture are analyzed.

A) Appearances (transparency and gelling tendency) are visually observed,

Bl The shrinkage rate (percent decrease in volume) of the hydrogel 1s examined.

Cr The gel strength (maximum stress) is neasured using & measurement instrument. = Instrument: Rheo meter (CR-L0ODX) 70 ~ Conditions: measurement item (gel strength), penetration distance (2.5 mm), table speed (50 mm/mini, and adaptor (No. 1 @&1bmm).

DY Deqgradability is determined as the number of days during which the crosslinked mixture remains in the presence of collagenase. - Collagenase concentration: G.1 mg/ml in PES

BE) The water content {change in water content) of the crosslinked mixture is analyzed by comparing the weight after freeze drying with the weight after hydration of tne crosslinked mixture.

Hesults 1} Appearance (transparency) oo Tanle Lo @ o i < - = A he ee ee em AO £6 £4 AEP Em eng A Een mR RAE RR LER CRRA 181 eh EF oy 22 ee

Po . nn Fo em Tp Cea a en dd nd pm Lg dn anh AD an ge i

Gamma-ray cose (kKby) | Hyaluronic acid Chondroitin sulfate : [I die | we }

Po Poko [I i

Appearance - * opague, TF gemi-Lransparent, FFF pe +n, amy * kh wR A : TT errs oe be

Lransparent, **** transparent (yellowish) - [aN dpe em eye yf “ - + 3 re mg ge 4 cr = sae 2} Shrinkage rate (percent decreases in volume) = (total weight (gg) of gamma ray-irradiated sample) -

I : FE £0 em ym es to . RL FN co AT eg a pn ~ ir weight (gp of gel mass) / total weight (gg) of gamma ray- opm om my dA am de on Te * 0 irradiated sample ¥ 100

Mm - To

Palle 106

Shrinkage rate | Biopolymer mixed with collagen : i LOY g percent decrease in | { ]

Povo lume) : :

EE A peppy a mae mt a = pore A 8 11101 6 i em seem 2 £8 ee mf mmm = = 5 = 2 8 418 5.8 | 011 | mm = 1 8 2 mm mmm 5m 18 = L551 i £5 7315500 a

Gamma-ray dose (kKGy) Hyaluronic acid | Chondroitin sulfate tt etm mmm Ee mmr A AL 1A PLA Pd AA me ee eee AE ERAT CW 4 dE aE 1 A A ERE hte in 10 mont

LE P00 | 00 :

PY HE POL : : z

Poe Ion [SR t

Fa Poli. | Ai : me eee AAA 3 RR bre hs BA Ah hb sh mlobmmmmmnmmm es cesser bestia ates em hs hm fb le 1 Se 2 em me 2 Gel strength {maximum Stress)

To TIE Fa 3 oy Ty ad Lallne dd a - 5 . bog “© . - - - gw i

Gel strencih (maximum | Blopolvmer mixed with collagen i | ;

Pstress) {MH}

Gamma-ray dose (kGy) | Hyaluronic acid | Chondroinin sulfare pe A CO 1 pe Pd AAA AAAAR ALARA RA tenn mmo] [I | ZUR Pa : RU Co 4 | L303 Pon 4) Degradability (davs)

Talle 18

Aci iT +30 gm py prs od mm fm TS es fo elmape YET men Tos re vd seemed rad bby eee Tm ees vy

DESTAGADI LITY (OAays) | RIODOILYyner mixed With Collagen ee

I i i

Gamma-ray dose {(kGy) | Hyaluronic acid Chondroitin sulfate x bvw Loe [I ! ; tt AE Ak ££ re ££ = + ££ one oe 0 toe eet 1118 5b ££ 1 3 er ££ 1 1115 1 18551 eter] i :

CED poet [Ly

Degradability (days) —- * 1 day or less, ** 2 days 1% or less, *** 7 davs or lass, and **** more than 7 days 5 J Waray oT ET eT {oh anoge in iat oy comb ent = 2 Water Conant Cnands in Wa lar convent

Spe Ty oy oy gpd pe my be om el Tm mT can dy be de . - (welaght (gy of hydrated collagen gel ~ weight {gy of

Frm meld ml perm oon bd pe J mm] 1 mee 1 aes teh . -

Irecze-~aried CroIslinxed collagen gel } Wa LGiin { 5) } > £m gm 3 pe 3 mm] dr rend mem tT oom gpm treeze-~dried crossiinked nollagen gel “0 falbie 19

Pa £3 gmmmmemseemssssemsermtmeme meee eee eee ete ——————————— [EE py Fp pein om rer | TES mre wren me rs armed rad dv me TT mw gm powWat er wonLant {han ge | DAopoLyner miKed With Coliadgen i

I foray

PL water content, Dold) i

Laima ray aoa {Key CoMYa@Laranld asia PALIONArGLTLn sul Date i 33.3 52.0

P28 | 23.6 | 20.5 en c

L¥amore

I By de wa od ny ym wm ed Te Dogg mpd me 4 em FT ~

Fa. Material prepdred DYOOLYIa diati ng 4 mlxXTdre of collagen and a chemical compound with radiation > Purpose: To examine whetheary a mixture of collagen and a chemical compound can be Tovrmulated,

Method i 3 Hi - Lo A ra A mo F 20 TY

I 3% Coiiadgen 1.5 mixed with each of 3% JA

TR pep om Nery fT ee To LO qn rr ng to PR o Do silicone (Dow Corning, 77-9806), 3% glyeerin and 6% PRS ti] = vey md Gm Ym pm wn . oem 3 1 a Fou — w — . i ee TT Re ar Ty 1d and then crossiinked by gamma-rays (5 ky). Fhe PBS . i a Toe a -” ie . £2 33. nw bp poe pe Jy of ny J i“ component includes 2.8 mg of sodium phosphate and 7.6 mg i“ Eo. ey] 2 po ” i AS me Te en, oo — cpm nn of zodlium chloride per wml of the final volume.

I) ru \ or 3 mem] yp py pn pn A pm en I. TN em om | be ed 2) fhe physical properties of The crosslinked

Garde gg emo qe on ay om |p poem en eel mixture ave analyzed. 1a A) Appearances (transparency and gelling tendency) are visually observed. 2) The shrinkage rate (percent decrease in voluma) of the hydrogel 13 examined. a ee 1 md an yt [, Ch pe oe ov i Ie ev mer re

ON] fhe gel sirength {maximum stress) ILE measured oy “on -, oy nm "oy - + 1m gm dm pe A

A Using a measurement instrument.

Toma de rma oe Fey mest ese {TR SOT = Lhnstrument: RNOec meter (CR-5000X) . - Conditions: measurement dtem {gel strength), oo de mn de dp en EE RE “3 I [RY le pe J =e Sed penetration distance (2.5 mm), fable speed (50 mm/min), and adaptor (No. 1 &lbmm) .

J ~ 3a I ; ; ; - 25h Dy Degradapiliity 13 determined as the number of omg lye ve 4 i” ee dd - ee bon oe] rd es mr = A —, - 1 days during which the cressiinked mixture remains in the 5 nom es EE mm - oes presence of collagenase, oe TT my yo ma eg ye En pe a ed an TTT pay mT 1 or Ra - Collagenase concentration: O.1 mg/mL in PBS

BY The water oontant (mumance in walror content oF fur hier Wate Coanieni LONage 1h water content OF ys = crores a td nlree me wh ire em mr} tre ened Ley TY Er oT by 2 Giler CIOL iniRed maXuure LE oahalyZaea LY Conparindg LINED weight alter Ireeze drying with the weight after ured oy de 3 ema ey de Je pm men TO py be om Ley we hydration of the crosslinked mixture,

Results 7 Tym ms mw TY oe bor ry SYA TY i ApDodll dna Lransparandy

BE fe Ea TY

Tabiae 20

Appearances Chemical compound mixed with collagen !

EEBtlicone Glvesrin PERS ! meer em mem em ene mimesis ei ns sms sss sminmisnsomses {DT TINIE TL essessssstslos sonora memes se emeene eee eemmsmmemmenrerr emer i i

Gamma-ray dose bow * Fk LE i okGyv] !

Ls idee [

Ey

App raranos ( trans Parency ) - x Coague, rm Sel dee pe mo ty pe pT pe Ee oh Fen mn ~ 2m me mm em he ER Foe en Cpr ny wes my de transparent, wk transparent, dd Cransparent wren 1 err deh { YY 2 LL LOWLE

Z¥ Shrinkage rate (percent decrease in volume) = oe der mm de | og fe dae + my — I. A pn mer om pd A my Am ey od eo Su i (total weight (gy of gamma ray-irradiated sample) - wei ont (ey of gel mass } / total weidant (py of Gamma rayv-

FE LONT G)O0r gel mass total weignt JF Cn Gaming ray ong mye wen pn] mo oe nd i T TOY TY rrradiated sample X 100

Table 21

Shrinkage (percent | Chemical compound mixed with collagen ; 1 i i i decrease In volume) | Silicones | Glyrerin Long : rev me ee ee i 2 we hr Ad nb mb men mm em mmm | I SP —— rd 1h 31 Gel strength (maximum stress)

Tall 00

Ladle LL fone ar ron hy femme drm CC hemmd ermal emresitmed tnd seen nad tl mee om ren ve

Poe k ST DenGTn LEX Lm LL nem Cal COMpOunG mixXad With Jolladgen i

Pat ronal POL mms POY rar PRS ! ; i !

Gamma-ray dose (5 kGyy | - Pall IE en ee Tirso msn resort ete Te enstn tiene]

HI em wen odo mow Toa 3m oe 4) Degradability {devs} walilie 42

Gel strength {maximum | Chemical compound mized with collagen i oat rman Pod Yl em EO mmm ee dy Pome i

BU es3d Pon lL LLOone Poa YET IN EES i eRe ee mm pe i HE a emer bo : cee ! Cp bo [a

Gamma-ray dose (5 kKGyy jrEer joo EEE “5 ey ay de md TA Beg Fong em ; bi Dm Ty em Fe ~ I

Fav Leara ami Lit Y { Gays 3 - 7% ida Yon Less, TE £0 aa YS or less, TUFF days or Less, and TYE F more Than J days oY [HE a pre pm my dm pny [mdm me em vv wp deny we vy em ey A ws 3} We TE conuent (onange In WATE connent) = wre rhe “yf Rudrated mal lasoon cel RR - po (weight (gy of hydrated collagen gel - weight (gg) of

4 rrr oe od em mgm om gm om Tm Jemed men] me 7 eri ph ; oo freeze-dried crossiinked collagen gel) / weight {(g} of 4 wd an) ig men win on em a we em od cy pm UTE ne - 3 frecze-dried crosslinked collagen gel

Table Z4 fromm meet me mS ——————_—_—_———————_—

Water content {change Chemical compound mixed with collagen i . . A me om — TT eS i ino owaber content, fold) | 2iliconsa Glvyeerin IPERS

A — J sone] i i

Gamma-ray dose (5 zGyy 111.4 55.4 P1209 ~ Ta my ewes | ao

Exampole 4

I om mn Td vm Led pe A em TT om Cy on om pm de pe ep oars

Fo Crosslinking of collagen by electron rays

Parpose: To induce martzal crosslinking of collagen by a low dose of radiation. The partially crossiinked collagen 1s mixed with a gelling sclution,

Ll Try pm de my py nen po on ; Ce FS . 3 Io Cone ETT Te ye -

Lu and whether the mixture is gelled 1s examined as =z

Anam phe he pm o£ yn TT an ng po pe ng pe pn pm en — ~ i pd Ad Ae dm function of collagen concentration and radiation dose te a (FIGS, to 11).

Method = ~ [ig ce, on od "1 [ae — Toad men em oo LT ho] ™y ye iy 0.5 and 1.0% collagen solutions (pH 3.0) are vreparad.

Z2y 1.0% collagen is irradiated with electron rays a odpm em em im mo i A ™ o [— t I Te - at dosas or 0.1, G.5H ang 1.0 KG Yo. pe oom em Tm ere ey To Dr meld meme naa mF em 1 ome ~ vom

Fy 0.5% coliagen is lrradiated with electron rays ob dm on £0 om 1 Fe sb 2 Te Tes at aoses of U.bh, 1.0 and 2.0 kG Vo. 5 y 1 Pa opm ee A 1 i — ow Ge od am dee nd

ZU 4 } he appearances ol the electron ra Y-—ilrradliated samples ars compared. jo EF yn wm 4 om 1d vem mn gm en Do ae ~ on on To de pe I. 2am mm 5 The partially crossiinked solution 1s mixed with a gelling solution, and whether the mixture is gelied 18 opserved. yO te bh 4 on Trade 3 mpm ow a To wo TITY am TON wen T —~ £ SAT NT 250 Gelling solution: Z.2 g NaHCO: in 100 mL of 0O.05N

MTL EE med A pn oe

NaGhH and 200 mM HERES. no I my oe — oo ype Su jou Ye ee - 0 den - mown yy pr ont cp mam bY ee

A) The appearance of he Jeli 13 examined visually 3 OY wy te Toy pm rem to TTY men and by a Lpectrophctometer (£10 nm).

Bl The gel strength (maximum stress) 13 measured with a measurement ingtrument.

Table 25

PALER nance GL RIeCLICni- Tay Goss Cw i

AE : E : aed ion | :

mp 20 a [yr : a Aor [- Laon :

FConcentration 0. 0.5 P10 Eau : i i { ;

LE sen A AR 8 1 15 1 6 £1 1170 mtn ted ! 1 i i { yn ok [I a :

Pole iow * | we Dede wk :

L oa ny po Em Tad Sem em gn my pe np pe To de Fo

Eppearance of solution- * transparent solution, ** or Cy yr am rd cy Torah ee Den rem my emt rs eee de frp pepe =

Cransparent sclution (Lncreased viscosity), *** partial

A— - Ik kk ou mV de rp mp mass, and **** compiets mass 1} Appearance of gel {visual ochservaltion) on Mo Pe yo . ialble Lo

Apmearanas of {| Blectron-ray dose [kGy) : creed :

Concentration CLD REI 0.5 C10 L3G i ; i i i | | i !

Ton Powwow [EE dk ow ow Pos 5.5 Le Lo | ws Er Lo :

ApDearancsg oi gel: not gel led y *Eogemiotransparent gel, and *** opague gel. 2) Appearance of gel (spectrophotometer, 410 nm)

Table 27

Appearance of | BElectron-ray doze (KGy) : gel :

Ce pm nt Doon [os IP :

PLONOent ration Poly. pL. ELL i

Co

OE : : i § i i

P10 PEL 34 ; 2.22 Vile : : i 3 : { yom Pos mn i HR i

ULh LLG i= POLuY

TM

LAS

I ~~ 1 ov qe yp Re J. LR. FE eb 2) Gel strength (maximum stress)

Pel strength | Electron-ray dose (KG) i [maximum stress, | i : 13)

PO ye vam oe 1 IE Poy roa ~ oo

LOnCEnTIranion PLL ld LD i i | : (5)

Fyn PA Le ong [mot i

CLL FLO PO. ae | 0.11

PhLh COL O06 Po PALER bbe Eee A A 1 Ee A RRL AR 1 OT £1 2 2 be 1 £8 £1 £m £1 44m 2 2 mm = mm £m £m 2 £1 ££) 2 £m £m 2 2 4mm 4 4m mt mm em mmm mmm]

Exampie 7

Oo Fwaminarion of martial crosslinking of ecollaoen ra DEAMINAaTIOon OL Partial Crassiin kan oa OF OO LL gen by electron rays

I ov Fo wr [TR . ~TT A - oe

Purpose : Ne EATING whether Collagen 13 polymerized by lrradiation with electron rays.

Method

Ly 1.0% collagen 1s irradiated with elechbron rays at doses of O01 and 0.5% kGy. 2} Each of the samples is analyzed by HPLC under the following conditions.

Ay Instrument: Waters HPLC system

B) Column: Ultranhvdrogel 2503, 1000

C) Sample concentration: 0.1% (1 mg/ml) 1a D) Intection volume: 50 ul

BE} Flow rate: 1 mL / min

Fy Analysis time: 10 min

H. Hydrogel and recovery of radiation-crosslinked 12 collagen ard usability of radiaticn-crosslinked collagen for wound dressings, cosmetic products {mask packs) and the like.

Purpose: fo examine whether radiation-crosslinked collagen can form hydrogel and can form a film when being dried. Also, ta examine whether {he film formulation contains water and 1g racovered, This experiment is a basic experiment for applying radiation- crosslinked collagen in the form of hydrogel.

Mat hod 103% and 6% collagens are prepared in dishes. 2) Each of the collagens 1s irradiated with gamma- rays at deses of 5 oand 25 kGyv. 3) Collagen hydrogels are dried ar OOM

Cemperature. 4y The elasticity of the film is measured, and then the film is added to distilled water to form hydrogel, and the recovery vate of the water content therecf is measured.

Results yo 20 festa eee eee i i Porm mpm 1nd dens Fol ono bo ae ep dn pe i PELasticily Ob Water content i ; : film recovery rate

Po Co :

Pa i i" HI [I 1 iu 5% ky PoE wk OA 1 3% PS kGy Pan J i IE DORGY Fok Pau i [I on - Fa Por i

PEE 5 ky x RIL, : { a OCP SSE RS ! 25 kGy * Pad :

FF mae RE 4 tt 1 A115 £5 0 om ee ee pp 4 te 25 PA AS 11 PL 180155 7 PARAS AL LALA me} oT dv dey oy LEY py ££ JR — 4 my pe po +

Elasticity of film formulation * broken, * ny ye EEE 4 hard, and pent

TT oe py bre om VTE En i ay Fld ers ae —

Water content recovery rate (%) = {thickness (mm) on, AT vq oo de ~ img Tr gn en ge Li en pond Ge yp] ue og ey] ; =m] . nn . 4 AT Tou gx em] pe ~~ 3 of water-absorbed hydrogel / thickness (mm) of hydrogel before drying

Fawamm io 0

LXallioise =

T TT om pm o£ wm lA an de Cs em my md - fmm 3d im een en em mp om oy A Ge

I. Use of radlatlion-radiated collagen as grafico mMaitariad

Tha a Fe py o pay 1 oy vq em ry wn en di dng Te ew on Jem fm yp pny ged

Purpose; [ae EXamLne wheather particieaes motained £ - = od me oe pe moon bo pe Le em ed It Fv 3 LD pn preg og } am de a - omy Fy pn i Lrom a radigtion-crossiinked hydrogel formulation can be

Tyo ety or im A ri my de ne Se Ee asad as graf matelials.,

Bie Te md

Met hod yA radistion-orossliinked hydrogel formulation is

Cmte spd En mm be grees re dem ee

GUOUnG Wit oa homogenizer.

TE, SA mY Tom wp od ye ep my § - yp pg vw od 17 dem pe or Foy om Le TE ur vy oy

LD a} ine OYarodeal 15 QYrouna THESE le DOL LOWING

COnGdLTiIOoONS. 7 ha EE oy An TI morro on Lem em am TIT ry od mp A sa Td ge

AY Instrument: H omodgenl ier {IKA T25 di SrTal uLtra- der yy ope gn y

CUT rIax) = [EERE gp rye Ed ey = AOA 0 pb vi Ty

B} Grinding conditions: rpm = 4.0-240.0, 1/min 5 L WO)

Ld ALU 3 Te PTE RI o nm a1 J 2 gm a sm ym - pm gd 2) ihe particles are fiiied Into a 5 VILnge, ano ham 4 pr mm med Ty ean be ves vessel En Ye pn em gpa pm gee pm then inhedied Through the neeGle CL The SYIXInge.

TI pene we

ExXampae Lu

T = ~ vy oon 1 pe oe om Tee oh 3 de oo amy A my eed my Ae py me em] eb 3

J. Thermal stability of radiation-crosslinked a Cob laden 21 rpose:d to examine whether radiatlion-crosslinked me TT nem 4 Fo ey NE dey TUE . de en en gee on TY Foe adm E collagen 13 thermally stable. After thermally treating

The collagen, whether the collagen maintains the properties of hydrogel 1s also examined.

Wim +o. om atic] toad eer Dodd yey

Material and test conditions

LY Material: 3% collagen (ph 3.0}.

ER Croaae linking yr el ed ey en Fee TE ay een en cen Hy ee a) LHOSS LINKING CONGLT LONE. Col Laden SCLUT ION, crosslinking bv ooamma-rave at doses of Bboy DE erly red

LLODs LLR LLG DY gamia-oays: dn acses O00 L HKuayY, £2 KY and [og Ay fe

J 40 kG Yo. - Tim oy A ae me i tae 2 2 a oa a oo pn oo 4) Heat-treatment conditions: 40, ol ancl HO:

Fem te rE aT Ie foo oe J bo pom yen neat-Tregiiment Dims: 1 Nou.

Method 153 Badiation—croasiinked coll aman Nasri nag = ] FAC at ion - Crosslinked COLiagen nasindg a

Tn ard mie doa J Co ene py £3 crn A et bY mn ae ne of Ie

LA SULTOADLe LTE 15 oredara a SC Lhe Wel ght Cres ro i 18 measured,

TH Phe evar rear] ora a bod rm demrt een TY mem doer pm bom ened pe 27 The prepared crossiinked collagen 1s placed in oy STE mT - oo ot eno ten or : IRR + om de £ 5 me be on = Fen a ConTalhelrl and peated Ln oa walter Daltnh Lo each ol Lhe oF Tren Err ae mts ms a meh hm

Ca hd LT LZ nA LL LE RID Load AL FALE

To TNT A me dm me de w . vp een med taps a eve NF Eh

J 3) Alter heating, Lhe appearances and welgnit on Lhe em TE meres Pp rn pened collagen Jes are examined. 4) The changes in weight caused by heat breatment

S. ne me A ] TT end eT ib de ge am mn am nn TE en de TTT mee

LE examined. Rowavwey, Whether nNon-grossiinxeda Collagen en Sm tr en ed . mm re drm bem meme — meen TTA 5m mn Ted 18 Ggnatuired 13 aertermined Dased on galling LI COL

ZL Temperatures COonairtiLons.

Domed diem] mm ( TAN wf {dea Eom cen eee

Raz = Lauda LL Eogrilud Lo } ha ALS oR REA Led Wie A nT wor oy pep den om en — do qe mm dn oy pm Ae 4 ao 4 7 [Rp ode dk FM weignt aiter heat treatment) / initial weight TOO) r Tim mn be boy am Ae mem sie my mY Tn URE bo Purple my de pod 5y Whether Lhe delormed Collagen gel 15 nydarated

Ye mera LA men brim tern 4 oe ries Ermer ngs oem " eXaminod, Ihe nydralt lon 18 periormed Ln water Loy op a i) and 74 hours,

Tm be my pe rE py wm EE = Bo — am oy Emp

Water content recovery rate {%) = weight after

Froped oom 4 Pmt Ed mt agra d Em Emel oa Wr emeesl * TON deat ion / initial Welght on Ccoilagen in YT GOs Lou J on Tent ep d= Fm CYNE LS DPS TOE rE Ta dem vem een ry mt yn hr ee ren

OJ Wino nen Lhe appearance of WaLer-Containing gel

Ta ohm TL Soo ao Tar gms yr Tory gad

LH Changed at oy i) Lame M andl ied,

D6 Themen] oe

BY mESUITES

Toe 3m Tom hea

Lalhise ou pag | Appearance [Residual | Recovery | Recovery I Remarks : : on de om Tor ny mdm | ho dee FT | en ot = i wn dr { i i | atter hear | rate (%) | rave (%, 11 rate 1%, | : i : | i ‘ : TreATment | nrg i 24 hi) i ee eee 12m a a eee i ee eee i i . } i i i ; Ls amma ar Lo I _ Dem TY meme ! bo. PV LECOSLTY ; : | i Lo liaoen i

HORLY l .

I decreased : ! | denatured ;

Fd f DL : r i Transparent ! | ; | !

Fo Weiler i x Poa on ion oo Loran a { : 3 KY i } i Pha, La. Poles i i ! cshrunk gel | { ; : i ¢ 3 : + i i H— i i i i : | Transparent i i ‘ : i

Pov gms Ea i - Torey on O70 Form i i

PA KGy PAL wi, poLbESLH i i { Jd . 5 i | i : : [Transparent : | :

Van bres | i HT oh foe t i pal Ky ] Pid. Vola Pola Ld : : ; Cahrunk gel | | i :

Tomes Tom 21 faple Si

Fp nm At renee ry

Coan I, mn dda t HE Tan pn nae gy PTY een ae be i

Lomb C Lo ADTERa range PoMEeRE paula l Po Regovery | mECOvery folema rks i : Latta haat orate (83 Povate | Ge Tl orate [i : ; ; Patter neat borate (uw) porate {w, 1 vale (5, i ; ! lt rantment | Pom ms poy ; i

Lona Lite i [EEA [A SE A i i ; Viscosity iP - b- Po Oo lagen !

PM Tar | - i ! ; : = i

Pol RY ‘ i i | ; ] = Uden vim ma ed : ! | Pormrmm dy ved i poe Crease] i i | Plena rayed 3 i i i i i i i om perme Om mer Pn : § ! brim vn cx de rn eo i

ID Koby Pl lexibie i - Po [i Pens tured i

Eo et 1 A 1 5 i 1 AA AAA AA rescence

Un i : i ; Plransoarent i t i

PTI Lee | + Peon on A oe | i

Loan RLY . Pos Bi, 4 Gon, i fehrunk as) : ! f : i Plransparent i i { i

ATT ler - Poy oem Pmnaon ooA { i

SLU KY ! SZ fra Dd Ln | i shrunk gel : | ; eb to 30

Table 32 50 C ADDBATANTS | Hams cia lL PREC ove ry Mextoyea ry | Hemarks :

SITE Meat rate {Fj porate ti, Il rae a, | i ! | : : | : : pm pe | Dot Doria ges i i

DOL Yemen yg | PE | «<4 hin | : re re care eae 11 1m 1 15 A £1 A 2 2 et 8 18 £8 £1 ££ £8 ££ £2 ££ mm £££ ee eet eee em =A 541112 48 At. 214028 mi =A mm A eee me 55 AS AB ABA A AAA SHS + rmemrenod A. { ! | : i | i

Tosrd ppimm 5am | i | Fel TT a pen en 3

Poy mC L LY PT Po ~ PLD La adan i

Un nme I i : i : :

Io RAY i i : | ! i

Doren enn mend i { Cortera ty rad i

Glin read hed i i | Polson ad raed i pment te Zen ni rmbt foreman star ats ss nen i i :

Td ered ot i i Vom TT meg va i

BN Poaed AUG iT tT | - olla gen : 7 Bly | ; | J ] = : 3 PTs mm Es mn od i i i | Paenanured ] ea 110 rr oi EE E— a

Transparent i i ‘ me x Posen Pome Ty on a : £3 Koy | PAu Pola, pha :

I sbivsime oe] i i ! i i WHEL. dad IN Sd LA i i 3 i Transparent i } Eo } | :

I 24 kGy PAL 2 Pals | 80.6 ! shrunk gz I ; :

I ot pet 3

Table 33 - 2 ; i i i i aa Fp pen pm ge we dpm pe Th pT em Te Tmo lye | Tommmitroy vt DT en yy pe Porson mov bee ] mis C SOOT ALT LTH Appearance Residual Renova Y Re oovery Porc TRA = ! i - i } i !

Date a Thay Prenat vate 0%) Porm ga 4 Dent { i water aie heat CATE LE) TELE LE, rate (3, : : : treatment | CLohm Pad hm :

J R83 eR BRL A LR PLLA 8 RA 8 SE em errr een i per mpm een et embed t wee i TTA am pm cr doy | i Pre TT meg ae i : VAISOOS LLY - Po Hi PLOoLLagen : | decreased | ; : Cdenatured ee ett re him A A TTT a Fame mens toes I RE ——— erm eee ee trim A — 3 L , | ! i 3 - b ; Changes todo b= Po Collagen : { | Liguria | | i Pasnatured

LON Le | or mova ey bom A Poon © i ! i

Pas Ray ol Dansoarant PSD Pow D i : i ae

Tp -_________ ™ © i i i t i i y opm TF i i i i : 1 shrunk gel : i { : et ems ores es een etree ern i . 3 | Lransparent Lo : :

PAD KGy i ) i Catz Pend ; : Lshrunk gel ; ! : aul ny yet ~ + J em oem my Tom — de . py pr Ae To pe md ££

The presont CLC 0osure relates TO & me Tod Tor - eT 2 1, - .. TN er a A] am de 2 —- 1 om 2 qn pm en td -

CrOSsSLInKLINg collagen by radiaclon and aims To provide a

TY oa 3+ a rad ped 5 . Em ram [PR mmo coliagen hydrogel which is used for wound dressings and - ¥ - “1 ro oy £7 b ot 1a] a my - ced : ; one A. or gralt materials. The crosslinking is carried out at room

Cemperature. mn vd Fd my DT a oy ppp pe od pm eA Flom wm mm en A eS men | pe eng ve

Spe QIrLcally, JACCoOrdlng Lo tne present disclosure /

Yo on ce aed eon mn Te be od mT mre yg] Ue yen od 3 ine radiguvlion-crosslinked COL laden ger i 1s rsaed Ln

VaAarlous manners as IroLlows.

Tn Th a » a fo mo . pn qm iN 4 TT es 11 Frrst, a5 SIOWnD 1h FTG. a. & partially crosslinked colla en chained Ws LOG a Low g0nsa [a mm mb ep 5a i. A de om ye 1A + +e A REE radiation 1s poured into & mola together wilh a gelling cpm Taq mn mE ee sees Sa Te momo md rT meen ems am ed

SCLUTILON TO norm oa gal. Lells and a medium are pourea on the gel, and ithe cells are culfured on the gel. The

TE EE ode mm dm] Tyrrel vem bY 23 Free remedy om nd ned es

Lo cultured material {hydro Ciel may be used Ln a mixture rio - \ I vp yp pm ne Tad - en pn bhp

Wino oa Cosmetic component, OY mixed with a cosmetic component TO Droviddse 4 MASK mac <, OY used 35 3 WOUNnG ym Cd - Tam mpm 1 dome doe mem oh ge Poel dom -. rovesed moe om dressing, Or 135 applied TO an inured skin, or used as a

SKIN Jrart Materiads. re ox ~ J a. im pm n vy Fane. mT 50 i. ye mete 4 ew TE xa

ZU Second, AE SHOWn 11 FRE 15, a partiaily 12m been od NE . Trobe om dem od ed - 3 1 -

Crosslinked Collagen ontained Lsing a LOW Ona [2

TAL ATION 1s maxed wiih 3 gel lL lng Solution To rorm a " 3 5 : Joe de md Ep FS op ATT Lose rb 4 em gel. The gel 1s dyled to form a thin film, after which e o ees b Te i meld ram mores pve read ey £37 pp wml Aden mem DE oo

Cells anc a megium are poured on the iim, and the Cells

SR soe m1 Rie med vm Fhe £4 Ten Ten ey Toby 3 pepe 1 meen dT en vemoar £3 are cultured on The iim. Lhe Culture corneal Cells may

Tvs rm From dem moe egies]

De graried LnUo an aeveballd. a : en co To mv oe oT le -

Snirda, 83 SOW in Ei. iy a PArTI&ELLY mcr cr 4 dren od em TT mrs mT nm yen rpm os T ye Ame ~~ F

Crosslinked CoOL Lagan ontained US Lng f= 20W Gana Of cm A med ey dm Spam nad ee om TU mer een Dade deny mmed eee 1 Te radiation 18 mi¥ed with Aa gelling scLution and oeiis oF i ’ - I ate now Sm a ere Th ~~ TFrrrmed mst NE NP and the mixture is cultured. The cultured material may en fr eiirne Fore Tl meget mom am 1 er cored pre Fo mel py em pe ground finely, placed in a syringe, and grafted into

The skin or hone Dy Ine on.

Herein, the gelling solution that is used 1n the present disclozure is preferably a mixture of sodium hydrogen carbonate {(NadlO.), sodium hydroxide (NaOH! and

HEPES.

Industrial Applicapility

The radiation-crosslinked collagen cel according ey hed bs fy To ear Aes fa] on or a = h TESTIS TA hm 1 Tien io i ™y ol

TO Lhe present disClOosure, ang Lhe Dreparation ne TNO ana use thereof sho substantially reproducible amy] lta and use thereci Snow Substantia YVoreprodueciole resilliis.,

Particularly, when the present disclosure is carried out, At can contribute to the development of industry, and Thus should be protected under the patent Law.

Claims (11)

1. Claims i. A me £ hood Tor Creparing a radiation crosslinked collagen gel, the mes nod comprising Lo irradiating liguid collagen with radiation to prepare a crosslinked collagen.
2. zZ. Tha method of claim 1, wherein the radiation is any one selected from among gamma-rays, @lectron rays LG and Yeravys,
3. 3. The method of Claim i, wherain The concentration of the collagen in the liguid collagen is

   0.00% (W/V, and the dose {dose rate x time) of the radiation is U.1-40 kGy/hr.
4. 4. The method of claim 1, wherein the method comprises mixing 1% (W/V) of collagen with each of 3% (W/W: of synthetic polymer Pluronic F-127, 1% (W/V) of FEO (polyethylene oxide (MW. = 100,000) and 3% (W/V) of hydroxyapatite and crosslinking the mixture by irradiation with gamma-rays.
5. 5. The method of claim 1, wherein the merhod comprises mixing 3% (W/V) of collagen with each of 33% (W/Vy of pilopoclymer hyaluronic acid (M.W. = 2,000 Ky) and 3% {W/V of chondroitin sulfate and crosslinking the mixture by irradiation with gamma-rays.
6. 6. The method of ¢laim 1, wherein the method comprises mixing 20% (W/V) of collagen with each of 3% (W/V) of silicone (Dow Corning, 77-9800), &n (W/V) of glycerin and 6% (W/V) of PBS and crosslinking the mixture by Zrradiation with gamma-rays, in which the PBS component includes 2.8 mg of sodium phosphate and 7.6 mg of sodium chioride pay mi of the final volume,
7. 7. A radliation~crosslinked collagen gel prepared by the method of claim 1. o wt a.
8. A method of using a radiation-orossiinked collagen gel, the method comprising: obtaining a partially crosslinked collagen using a low dose of radiation; pouring the partially orossiinked collagen together with a gelling solution into a mold to form a gel; pouring cells and 2a medium on the gel; culturing the cells on the gel; and using the cultured material (hydrogel) in a mixture with a cosmetic component, or using the hydrogel a3 a mask pack Ln a mixture with a LE cosmetic component, or using the hydrogel as a wound dressing, or applying the hydrogel fo an injured skin, or using the hydrogel as a skin graft material,
9. 3. A method of using a radiation-crosslinked 23 collagen gel, the method comprising: chialning a partially crossiinked collagen using a low dose of radiation; mixing the partially crosslinked collagen with a gelling solution te form a gel; drying the gel fo form a thin fllm; pouring cells and a medium onto he 2% film; culturing the cells on the film; and grafting the cultured corneal cells into an eyeball.
10. 10. A method of using a radiation-crosslinked collagen gel, the method comprising: obtaining a partially crosslinked collagen using a low dose of radiation; mixing the partially crosslinked coollagen with a gelling solution and cells; culturing the mixture; finely grinding the cultured material; placing the ground material in a syrings; and grafting the material in the syringe into the skin or Dong by injection.
11. 11. The method of any one of «laims 8 to 11, wherein the gelling solution comprises a mixture of sodium hydrogen carbonate (NaHCO), sodium hydroxide (NaOH) and HERPES.